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Dihydropyrimidine dehydrogenase (DPD; DPYD gene) variants have emerged as reliable predictors of adverse toxicity to the chemotherapy agent 5-fluorouracil (5-FU). The intronic DPYD variant rs75017182 has been recently suggested to promote alternative splicing of DPYD. However, both the extent of alternative splicing and the true contribution of rs75017182 to DPD function remain unclear. In the present study we quantified alternative splicing and DPD enzyme activity in rs75017182 carriers utilizing healthy volunteer specimens from the Mayo Clinic Biobank. Although the alternatively spliced transcript was uniquely detected in rs75017182 carriers, canonically spliced DPYD levels were only reduced by 30% (p=2.8×10−6) relative to controls. Similarly, DPD enzyme function was reduced by 35% (p=0.025). Carriers of the well-studied toxicity-associated variant rs67376798 displayed similar reductions in DPD activity (31% reduction). The modest effects on splicing and function suggest that rs75017182 may have clinical utility as a predictor of 5-FU toxicity similar to rs67376798.

Initiation of DNA replication in eukaryotes is exquisitely regulated to ensure that DNA replication occurs exactly once in each cell division. A conserved and essential step for the initiation of eukaryotic DNA replication is the loading of the mini-chromosome maintenance 2–7 (MCM2–7) helicase onto chromatin at replication origins by Cdt1. To elucidate the molecular mechanism of this event, we determined the structure of the human Cdt1-Mcm6 binding domains, the Cdt1(410–440)/MCM6(708–821) complex by NMR. Our structural and site-directed mutagenesis studies showed that charge complementarity is a key determinant for the specific interaction between Cdt1 and Mcm2–7. When this interaction was interrupted by alanine substitutions of the conserved interacting residues, the corresponding yeast Cdt1 and Mcm6 mutants were defective in DNA replication and the chromatin loading of Mcm2, resulting in cell death. Having shown that Cdt1 and Mcm6 interact through their C-termini, and knowing that Cdt1 is tethered to Orc6 during the loading of MCM2–7, our results suggest that the MCM2–7 hexamer is loaded with its C terminal end facing the ORC complex. These results provide a structural basis for the Cdt1-mediated MCM2–7 chromatin loading.

Highlights d ORC self-interacts and dimerizes at the M-to-G 1 transition and de-dimerizes in S phase d ORC dimers provide symmetric platforms to load the symmetric pre-RCs at origins d Upon ORC de-dimerization, each single hexamer binds one of the two nascent origins d ORC dimerization is essential for and regulates pre-RC formation and DNA replication

Clinical studies have identified specific genetic variants in dihydropyrimidine dehydrogenase (DPD, DPYD gene) as predictors of severe adverse toxicity to the commonly used chemotherapeutic 5-fluorouracil; however, these studies have focused on European and European-American populations. Our lab recently demonstrated that additional variants in non-European haplotypes are predictive of 5-FU toxicity. The objective of this study was to identify potential risk variants in an under-studied East African population relevant to our institution’s catchment area. The DPYD protein-coding region was sequenced in 588 individuals of Somali or Kenyan ancestry living in central/southeast Minnesota. Twelve novel non-synonymous variants were identified, seven of which significantly decreased DPD activity in vitro. The commonly reported toxicity-associated variants *2A, D949V, and I560S were not detected in any individuals. Overall, this study demonstrates a critical limitation in our knowledge of pharmacogenetic predictors of 5-FU toxicity, which has been based on clinical studies conducted in populations of limited diversity.

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex.

SummaryRegulation of DNA replication initiation is essential for the faithful inheritance of genetic information. Replication initiation is a multi-step process involving many factors including ORC, Cdt1p, Mcm2-7p and other proteins that bind to replication origins to form a pre-replicative complex (pre-RC). As a prerequisite for pre-RC assembly, Cdt1p and the Mcm2-7p heterohexameric complex accumulate in the nucleus in G1 phase in an interdependent manner in budding yeast. However, the nature of this interdependence is not clear, nor is it known whether Cdt1p is required for the assembly of the MCM complex. In this study, we provide the first evidence that Cdt1p, through its interaction with Mcm6p with the C-terminal regions of the two proteins, is crucial for the formation of the MCM complex in both the cytoplasm and nucleoplasm. We demonstrate that disruption of the interaction between Cdt1p and Mcm6p prevents the formation of the MCM complex, excludes Mcm2-7p from the nucleus, and inhibits pre-RC assembly and DNA replication. Our findings suggest a function for Cdt1p in promoting the assembly of the MCM complex and maintaining its integrity by interacting with Mcm6p.

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